Figure S4. Distinct binding properties... | Rockefeller University Press

Figure S4.

$Distinct binding properties of LMX1A and LMX1B for ATG8 proteins in cytosol and nucleus. (A) GFP-TRAP IP of nuclear and cytosolic fractions from lysates of HEK293T cells expressing GFP-LC3B immunoblotted with anti-LMX1B antiserum. A faint band at the correct size for native LMX1B (arrow) is detected only in the nuclear fraction for the GFP-LC3B pull-down. (B) HEK293T cells expressing GFP-ATG8 proteins and LMX1B-FLAG were separated into nuclear and cytoplasmic fractions which were subjected to GFP-TRAP. Interactions with ATG8 family members were detected only in the nucleus. Immunoblotting for p62 is included as a positive control for binding. Fractionation is demonstrated to the left using Lamin B and GAPDH as markers for nuclei and cytosol, respectively. (C) Domain schematic of human LMX1A and LMX1B showing the position of a possible LIR motif in LMX1A (left). Alignment of the putative LMX1A LIR in different species (right). (D) GFP-TRAP co-precipitation of LMX1A with GFP-ATG8 family members in HEK293T cells. 5% protein lysate from equivalent GFP-expressing cells is shown as “input.” Immunoblotting for p62 is included as a positive control for binding. Arrow indicates the position of the LMX1A band. (E) GFP-TRAP co-precipitation of LMX1A with wild-type of LIR docking mutant (F52A/L53A) GFP-LC3B in HEK293T cells. 5% of protein lysates were used as control for protein expression (inputs). Immunoblotting for p62 is included as a positive control for binding. Immunoblotting for p62 is included as a positive control for binding. Arrow indicates position of the LMX1A band. (F) GFP-TRAP IP of nuclear and cytosolic fractions from lysates of HEK293T cells co-expressing GFP-LC3A, B, C and LMX1A under basal conditions. Immunoblotting for p62 is included as a positive control for binding. Arrow indicates position of the LMX1A band. (G) GFP-TRAP IP of nuclear and cytosolic fractions from lysates of HEK293T cells co-expressing GFP-LC3B and LMX1A. Comparisons of pull-downs in full nutrients following 2 h starvation. Immunoblotting for p62 is included as a positive control for binding. Arrow indicates position of the LMX1A band. (H) GFP-TRAP IP of LMX1A in HEK293T cells co-expressing wild-type, acetylation-deficient (K49R/K51R), and acetylation mimic (K49Q/K51Q) GFP-LC3B. Immunoblotting for p62 is included as a positive control for binding. Arrow indicates position of the LMX1A band. (I) GFP-TRAP pull-downs in lysates of HEK293T cells expressing LIR mutant (Y290A/L293A) LMX1A and GFP-LC3B. Immunoblotting for p62 is included as a positive control for binding. Arrow indicates position of the LMX1A band. (A, B, D–I) Molecular weight markers are shown in kD. PSSM, Position Specific Scoring Matrix.$

Distinct binding properties of LMX1A and LMX1B for ATG8 proteins in cytosol and nucleus. (A) GFP-TRAP IP of nuclear and cytosolic fractions from lysates of HEK293T cells expressing GFP-LC3B immunoblotted with anti-LMX1B antiserum. A faint band at the correct size for native LMX1B (arrow) is detected only in the nuclear fraction for the GFP-LC3B pull-down. (B) HEK293T cells expressing GFP-ATG8 proteins and LMX1B-FLAG were separated into nuclear and cytoplasmic fractions which were subjected to GFP-TRAP. Interactions with ATG8 family members were detected only in the nucleus. Immunoblotting for p62 is included as a positive control for binding. Fractionation is demonstrated to the left using Lamin B and GAPDH as markers for nuclei and cytosol, respectively. (C) Domain schematic of human LMX1A and LMX1B showing the position of a possible LIR motif in LMX1A (left). Alignment of the putative LMX1A LIR in different species (right). (D) GFP-TRAP co-precipitation of LMX1A with GFP-ATG8 family members in HEK293T cells. 5% protein lysate from equivalent GFP-expressing cells is shown as “input.” Immunoblotting for p62 is included as a positive control for binding. Arrow indicates the position of the LMX1A band. (E) GFP-TRAP co-precipitation of LMX1A with wild-type of LIR docking mutant (F52A/L53A) GFP-LC3B in HEK293T cells. 5% of protein lysates were used as control for protein expression (inputs). Immunoblotting for p62 is included as a positive control for binding. Immunoblotting for p62 is included as a positive control for binding. Arrow indicates position of the LMX1A band. (F) GFP-TRAP IP of nuclear and cytosolic fractions from lysates of HEK293T cells co-expressing GFP-LC3A, B, C and LMX1A under basal conditions. Immunoblotting for p62 is included as a positive control for binding. Arrow indicates position of the LMX1A band. (G) GFP-TRAP IP of nuclear and cytosolic fractions from lysates of HEK293T cells co-expressing GFP-LC3B and LMX1A. Comparisons of pull-downs in full nutrients following 2 h starvation. Immunoblotting for p62 is included as a positive control for binding. Arrow indicates position of the LMX1A band. (H) GFP-TRAP IP of LMX1A in HEK293T cells co-expressing wild-type, acetylation-deficient (K49R/K51R), and acetylation mimic (K49Q/K51Q) GFP-LC3B. Immunoblotting for p62 is included as a positive control for binding. Arrow indicates position of the LMX1A band. (I) GFP-TRAP pull-downs in lysates of HEK293T cells expressing LIR mutant (Y290A/L293A) LMX1A and GFP-LC3B. Immunoblotting for p62 is included as a positive control for binding. Arrow indicates position of the LMX1A band. (A, B, D–I) Molecular weight markers are shown in kD. PSSM, Position Specific Scoring Matrix.